The present invention relates to genetic elements that suppress the activities of the human immunodeficiency virus (HIV). In particular, the invention relates to polynucleotides isolated from the HIV-1 genome, methods for isolating and identifying such polynucleotides, and methods for using them for the protection of human cells against HIV infection and/or replication.
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neurological dysfunctions, neoplastic growth, and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Daigleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gp120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348).
HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into the cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). To date, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The later stages of HIV replication, which involve crucial virus-specific secondary processing of certain viral proteins, have also been examined as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs have been developed to inhibit this protease (Erickson, 1990, Science 249:527-533). However, the clinical utility of these candidate drugs is still in question.
The lack of a satisfactory treatment for AIDS has led investigators to gene therapy approaches. One form of gene therapy involves the use of genetically-engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. For instance, replication defective vectors have been designed to introduce cell growth inhibitory genes into host cells (WO 90/12087, Oct. 18, 1980). One strategy attempted by several groups involves the delivery of the herpes simplex virus type 1 thymidine kinase (tk) toxin gene. The tk gene product is toxic to mammalian cells only in the presence of nucleoside analogs, such as ganciclovir (Ventakash et al., 1990, Proc. Natl. Acad. Sci. USA 87: 8746-8750; Brady et al., 1994, Proc. Natl. Acad. Sci. USA 91: 365-369; WO 90/07936, Jul. 26, 1990). Diphtheria toxin gene has also been used, and the gene was placed under the control of cis-acting HIV regulatory sequences (U.S. Pat. No. 5,306,631, issued Apr. 26, 1994). Others have utilized replication incompetent mutants of HIV which have the potential to express an inhibitory gene product in the presence of HIV tat (WO 94/16060, Jul. 21, 1994).
Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Efforts to identify appropriate protective genes have, in large part, been based on an understanding of the molecular biology of HIV replication. A few examples of this approach are as follows.
The HIV-1 Rev gene encodes a protein that is necessary for the expression of full length HIV-1 transcripts in infected cells and the production of HIV-1 virions. Transfection with one Rev mutant known as RevM10 has been shown to protect the cells against HIV infection (Malim et al., 1992, J. Exp. Med. 176:1197; Bevec et al., 1992, Proc. Natl. Acad. Sci. USA 89:9870-74). Typically, the transfectants are resistant to HIV-1 infection for about 2 weeks from the time of inoculation before resistant variants appear (Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11581-85).
In addition, Rev function can be interfered with by producing an excess of the binding site of the Rev protein, termed Rev Response Element (RRE), which prevents the binding of Rev to RRE of viral transcripts. A xe2x80x9cdecoyxe2x80x9d which consisted of a chimeric RNA composed of an RRE and a tRNA prevented infection of cultured cells for a period of greater than about 40 days (Lee et al., 1994, J.Virology 68:8254-64).
Alternatively, fusion proteins capable of binding to viral env proteins have been made to prevent the production of HIV-1 virions. Examples include a fusion protein composed of CD4 and a lysosomal targeting protein procathepsin D, and an anti-env Fv which is secreted into the endoplasmic reticulum (Lin et al., WO 93/06216; Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-93).
Antisense polynucleotides have also been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP 594,881; and Chatterjee et al., 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The ribozyme approach to forming an HIV-1 resistant hematopoietic cell line has been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06; Yamada et al., 1994, Gene Therapy 1:38-45; Ho et al., WO 94/26877; and Cech and Sullenger, WO 95/13379).
Roninson et al. described a method for isolating genetic fragments from the HIV-1 genome capable of protecting a cell from HIV-1 infection (U.S. Pat. No. 5,217,889 and WO 92/07071). The method involves the preparation of an expression library known as a Random Fragment Expression (RFE) library that contains random sequence fragments of the HIV-1 genome. Gene fragments referred to as HIV-1 Genetic Suppressor Elements (HIV-1 GSE) are then selected from the RFE library following an extensive selection procedure. The selection step involves transfection of the RFE library into a cell line to which HIV-1 infection is normally cytotoxic. However, the low sensitivity of this selection step greatly limits the practical use of the procedure. Moreover, no specific GSE sequences were reported using this method that were capable of suppressing HIV-1 infection.
The present invention relates to specific HIV-derived polynucleotides herein referred to as GSE that suppress HIV infection and/or replication in human cells, methods for isolating and identifying such polynucleotides, and methods for using them in the prevention and treatment of HIV infection.
The invention is based, in part, on the Applicants"" discovery that nucleotide fragments can be isolated from the HIV-1 genome, based on their ability to suppress the activation of latent HIV-1 in a CD4+ cell line. In this connection, any cellular or viral marker associated with HIV replication can be used to monitor the activation of latent HIV. An example of such a marker is CD4, which is conveniently monitored by using a specific antibody. While the majority of the cells lose cell surface CD4 expression after induction of the virus from latency, the cells containing HIV-1 GSE retain CD4 expression. A number of novel HIV-1 GSE polynucleotides are selected on the basis of their ability to sustain CD4 expression by the induced cells, and several of such sequences are further shown to protect uninfected T cells from productive infection by HIV-1. The GSE may function in the form of an RNA product or protein product, both of which are within the scope of the invention.
A wide range of uses are encompassed by the invention, including but not limited to, AIDS treatment and prevention by transferring GSE into HIV-1-susceptible cell types. For example, GSE may be transferred into hematopoietic stem cells in vitro followed by their engraftment in an autologous, histocompatible or even histoincompatibile recipient. In an alternative embodiment of the invention, any cells susceptible to HIV infection may be directly transduced or transfected with GSE in vivo.